Project Summary l The body?s entire complement of blood and immune cells is produced via hematopoiesis, a process mediated by a population of hematopoietic stem cells (HSCs). Hematopoietic stem cell transplants (HSCTs), are a common therapeutic tool for treating malignant and compromised immune and blood systems but have large mortality rates post-transplant due in part to the inability of the transplanted cells to successfully home and engraft within the native niche. Inspired by HSCTs, there is an opportunity to develop tools to understand processes associated with microenvironmental regulation of HSC maintenance and hematopoietic differentiation. Primarily found in the body?s bone marrow, HSCs exist in temporally and spatially defined regions termed niches that provide key external cues that direct HSC response. These niches are comprised of mechanical cues, signaling gradients, and co-inhabiting niche cells, all of which combine to form a vastly complicated system with transitional zones that promote maintenance, activation, or migration of the inhabiting stem cells. Co-niche inhabiting mesenchymal stromal cells (MSCs) have been implicated in maintaining HSCs via direct cell-cell contact and secreted factors, and are significantly involved in remodeling of the surrounding extracellular matrix (ECM), altering the matrix landscape that the inhabiting HSCs experience. Such matrix remodeling would alter the balance of cell secreted biomolecular signals surrounding HSCs, notably HSC-mediated autocrine feedback vs. MSC-generated paracrine signals. Tissue engineering approaches offer an opportunity to study synergies between the dynamic remodeling of a biomaterial landscape and heterotypic cell-cell interactions mediated by factors secreted by niche-associated MSCs. To examine the influence of these microenvironmental signals on HSCs maintenance, HSCs and MSCs will be encapsulated in a series of gelatin hydrogels with defined poroelastic (mechanical, biotransport) properties. This project will first define HSC maintenance in biomaterial regimes characterized by the balance of autocrine vs. paracrine dominated signaling, dependent upon the diffusive properties of the gelatin hydrogel (Aim 1). As MSCs are involved in dynamic remodeling of the hydrogel matrix, we will subsequently dissect the dynamic contributions of autocrine and paracrine signaling on HSC maintenance in a remodeled hydrogel matrix (Aim 2). In so doing, we will develop a biomaterial platform to examine dynamic processes associated with HSC lodgment and maintenance within native marrow niches, and offer design motifs for ex vivo culture systems to promote maintenance of hematopoietic progenitors.